Fusion peptide therapeutic compositions   

The application claims priority under 35 U.S.C. §119(e) to U.S. Patent application Ser. No. 60/842,464, filed Sep. 6, 2006, incorporated by reference herein in its entirety.

FIELD OF THE INVENTION

The invention provides a new generation of therapeutic compositions, incorporating fusion proteins (FPs) including elastin-like peptides (ELPs) and peptide active therapeutic agents. The therapeutic compositions of the invention enable improved solubility, bioavailability or bio-unavailability, and half-life of the administered peptide active therapeutic agents to be achieved, as compared to corresponding compositions containing the same peptide active therapeutic agents without associated ELPs.

BACKGROUND OF THE INVENTION

The disclosures of U.S. Pat. No. 6,852,834, issued Feb. 8, 2005 in the name of Ashutosh Chilkoti for “FUSION PEPTIDES ISOLATABLE BY PHASE TRANSITION,” and U.S. patent application Ser. No. 11/053,100 filed Feb. 8, 2005 in the name of Ashutosh Chilkoti for “FUSION PEPTIDES ISOLATABLE BY PHASE TRANSITION,” are hereby incorporated herein by reference, in their respective entireties, for all purposes.

The aforementioned Chilkoti patent and patent application disclose genetically-encodable, environmentally-responsive fusion proteins comprising ELP peptides. Such fusion proteins exhibit unique physico-chemical and functional properties that can be modulated as a function of solution environment.

SUMMARY

The present invention relates broadly to fusion protein (FP) therapeutic compositions including elastin-like peptides (ELPs) and peptide active therapeutic agents.

In the FP therapeutic compositions of the invention, at least one peptide active therapeutic agent is coupled to one or more ELPs, e.g., being covalently bonded at an N- or C-terminus thereof, to achieve enhancement of the efficacy of the peptide active therapeutic agent(s), in relation to the corresponding therapeutic agent(s) alone. The peptide active therapeutic agent-ELP construct has enhanced efficacy in respect of any of various properties, such as solubility, bioavailability, bio-unavailability, therapeutic dose, resistance to proteolysis, half-life of the administered peptide active therapeutic agent, etc.

In another aspect, the invention relates to fusion gene constructs, including heterologous nucleotide sequences operably linked to an expression control element, e.g., a promoter of appropriate type, wherein the heterologous nucleotide sequences encode a fusion protein including at least one peptide active therapeutic agent coupled to at least one ELP.

In a further aspect, the invention relates to a method of enhancing efficacy of a peptide active therapeutic agent. The method includes coupling the peptide active therapeutic agent with at least one ELP to form a FP therapeutic composition, wherein the peptide active therapeutic agent in such FP therapeutic composition has enhanced efficacy, in relation to the peptide active therapeutic agent alone. In one aspect the enhanced efficacy is in vivo efficacy.

Another aspect of the invention relates to a method of treating a subject in need of a peptide active therapeutic agent, including administering to the patient a therapeutic composition including: (i) the peptide active therapeutic agent to coupled with at least one ELP, or (ii) a nucleotide sequence encoding a fusion protein including the peptide active therapeutic agent and at least one ELP, operably linked to an expression control element therefore.

In still another aspect, the invention relates to a therapeutic agent dose form, in which the therapeutic agent is conjugated with an ELP.

Various other aspects, features and embodiments of the invention will be more fully apparent from the ensuing disclosure and appended claims.

FIG. 1 is an SDS-PAGE gel showing expression of a 37 amino acid peptide, using the expression and purification methods of Example 1.

FIG. 2 is a graph confirming the purification of the peptides resulting from the methods of Example 1.

FIG. 3 is an SDS-PAGE gel showing the results of ITC purification of BFP, CAT and K1-3, as set forth in Example 6.

FIGS. 4A and 4B are graphs of the increase in turbidity as a function of temperature of each of the fusion constructs of Example 8 in PBS buffer.

FIG. 5 is graph illustrating the blood concentration time-course for 14C labeled ELP, as set forth in Example 9.

FIG. 6 is a graph showing biodistribution of 14C labeled ELP1-150 and ELP 2-160 in nude mice, as described in Example 10.

FIG. 7 is a graph showing biodistribution of 14C labeled ELP2-[V1A8G7-160] in nude mice, as described in Example 10.

DETAILED DESCRIPTION

The invention provides therapeutic compositions incorporating fusion proteins (FPs) including elastin-like peptides (ELPs) and peptide active therapeutic agents.

The therapeutic compositions of the invention enable increased efficacy of the peptide active therapeutic agent, e.g., improved solubility, bioavailability, bio-unavailability (where desired to avoid build up and/or toxicity, for example cardiotoxicity, etc.), half-life of the administered peptide active therapeutic agent, etc., to be achieved, as compared to corresponding compositions containing the same peptide active therapeutic agents without associated ELPs.

For ease of reference in the ensuing discussion, set out below are definitions of specific terms appearing in such discussion.

The term “protein” is used herein in a generic sense to include polypeptides of any length.

The term “peptide” as used herein is intended to be broadly construed as inclusive of polypeptides per se having molecular weights of up to about 10,000, as well as proteins having molecular weights of greater than about 10,000, wherein the molecular weights are number average molecular weights. In a specific aspect, peptides having from about 2 to about 100 amino acid residues are particularly preferred as peptide therapeutic active agents of the invention.

As used herein, the term “coupled” means that the specified moieties are either directly covalently bonded to one another, or indirectly covalently joined to one another through an intervening moiety or moieties, such as a bridge, spacer, or linkage moiety or moieties, or they are non-covalently coupled to one another, e.g., by hydrogen bonding, ionic bonding, Van der Waals forces, etc.

As used herein, the term “half-life” means the period of time that is required for a 50% diminution of bioactivity of the active agent to occur. Such term is to be contrasted with “persistence,” which is the overall temporal duration of the active agent in the body, and “rate of clearance” as being a dynamically changing variable that may or may not be correlative with the numerical values of half-life and persistence.

The word “transform” is broadly used herein to refer to introduction of an exogenous polynucleotide sequence into a prokaryotic or eukaryotic cell by any means known in the art (including, for example, direct transmission of a polynucleotide sequence from a cell or virus particle as well as transmission by infective virus particles), resulting in a permanent or temporary alteration of genotype in an immortal or non-immortal cell line.

The term “functional equivalent” is used herein to refer to a protein that is an active analog, derivative, fragment, truncation isoform or the like of a native protein. A polypeptide is active when it retains some or all of the biological activity of the corresponding native polypeptide.

As used herein, “pharmaceutically acceptable” component (such as a salt, carrier, excipient or diluent) of a formulation according to the present invention is a component which (1) is compatible with the other ingredients of the formulation in that it can be combined with the FPs of the present invention without eliminating the biological activity of the FPs; and (2) is suitable for use with animals (including humans) without undue adverse side effects (such as toxicity, irritation, and allergic response). Side effects are “undue” when their risk outweighs the benefit provided by the pharmaceutical composition. Examples of pharmaceutically acceptable components include, without limitation, any of the standard pharmaceutical carriers such as phosphate buffered saline solutions, water, emulsions such as oil/water emulsions, microemulsions and various types of wetting agents.

As used herein, the term “native” used in reference to a protein indicates that the protein has the amino acid sequence of the corresponding protein as found in nature.

As used herein, the term “spacer” refers to any moiety that may be interposed between the ELP and the peptide active therapeutic agent in a given ELP/peptide active therapeutic agent construct. For example, the spacer may be a divalent group that is covalently bonded at one terminus to the ELP, and covalently bonded at the other terminus to the peptide active therapeutic agent. The ELP/peptide active therapeutic agent construct therefore is open to the inclusion of any additional chemical structure that does not preclude the efficacy of the ELP/peptide active therapeutic agent construct for its intended purpose. The spacer may for example be a protease-sensitive spacer moiety that is provided to control the pharmacokinetics of the ELP/peptide active therapeutic agent construct, or it may be a protease-insensitive ELP/peptide active therapeutic agent construct.

Fusion protein (FP) therapeutic compositions of the invention at least one elastin-like peptide (ELP) coupled with at least one peptide active therapeutic agent. The ELP and peptide active therapeutic agent components of the composition may be coupled with one another in any suitable manner, including covalent bonding, ionic bonding, associative bonding, complexation, or any other coupling modality that is effective to aggregate the ELP and peptide active therapeutic agent components, so that the peptide active therapeutic agent is efficacious for its intended purpose, and so that the presence of the coupled ELP enhances the peptide active therapeutic agent in the composition in some functional, therapeutic or physiological aspect, so that it is more efficacious than the peptide active therapeutic agent alone.

Thus, the ELP-coupled peptide active therapeutic agent in the FP therapeutic composition may be enhanced in any other properties, e.g., its bioavailability, bio-unavailability, therapeutic dose, formulation compatibility, resistance to proteolysis or other degradative modalities, solubility, half-life or other measure of persistence in the body subsequent to administration, rate of clearance from the body subsequent to administration, etc.

In the FP therapeutic compositions of the invention, at least one peptide active therapeutic agent is coupled to one or more ELPs, e.g., being covalently bonded at an N- or C-terminus thereof, to achieve enhancement of the efficacy of the peptide active therapeutic agent(s), in relation to the corresponding therapeutic agent(s) alone.

The FP therapeutic compositions of the invention may be therapeutically administered directly, or otherwise be produced in vivo from corresponding fusion gene constructs, including heterologous nucleotide sequences operably linked to an expression control element, e.g., a promoter of appropriate type, wherein the heterologous nucleotide sequences encode a fusion protein including at least one peptide active therapeutic agent coupled to at least one ELP.

The invention enables the enhancement of the efficacy of a peptide active therapeutic agent, e.g., by coupling the peptide active therapeutic agent with at least one ELP to form a FP therapeutic composition, wherein the peptide active therapeutic agent in such FP therapeutic composition has enhanced efficacy in relation to the peptide active therapeutic agent alone.

The invention may be practiced using any suitable therapeutic dose form including at least one peptide active therapeutic agent, coupled with at least one ELP.

The invention enables stabilization of a peptide active therapeutic agent against proteolytic degradation, by coupling such agent with at least one ELP to form a FP therapeutic composition.

The FP therapeutic composition of the invention may include one or more ELP species, and one or more peptide active therapeutic agents. As indicated hereinabove, the ELP species and peptide active therapeutic agents may be coupled directly with one another, or alternatively such coupling may be effected in a construct including a spacer moiety intermediate the ELP and the peptide active therapeutic agent.

The ELP species used in the FP therapeutic composition of the invention may be of any suitable type. ELPs are repeating peptide sequences that have been found to exist in the elastin protein. Among these repeating peptide sequences are polytetra-, polypenta-, polyhexa-, polyhepta-, polyocta, and polynonapeptides.

ELPs undergo a reversible inverse temperature transition. They are structurally disordered and highly soluble in water below a transition temperature (Tt), but exhibit a sharp (2-3° C. range) disorder-to-order phase transition when the temperature is raised above Tt, leading to desolvation and aggregation of the polypeptides. The ELP aggregates, when reaching sufficient size, can be readily removed and isolated from solution by centrifugation. Such phase transition is reversible, and isolated ELP aggregates can be completely resolubilized in buffer solution when the temperature is returned below the Tt of the ELPs.

In the practice of the present invention, the ELPs species functions to stabilize or otherwise improve the peptide active therapeutic agent in the therapeutic composition. Subsequent to administration of the coupled peptide active therapeutic agent-ELP construct to the patient in need of the peptide therapeutic agent, the peptide active therapeutic agent and the ELP remain coupled with one another while the peptide active therapeutic agent is therapeutically active, e.g., for treatment or prophylaxis of a disease state or physiological condition, or other therapeutic intervention.

For example, the ELPs in therapeutic compositions of the present invention may comprise ELPs formed of polymeric or oligomeric repeats of various characteristic tetra-, penta-, hexa-, hepta-, octa-, and nonapeptides, including but not limited to:

(SEQ ID NO: 1) (a) tetrapeptide Val-Pro-Gly-Gly, or VPGG; (SEQ ID NO: 2) (b) tetrapeptide Ile-Pro-Gly-Gly, or IPGG; (SEQ ID NO: 3) (c) pentapeptide Val-Pro-Gly-X-Gly, or VPGXG, wherein X is any natural or non- natural amino acid residue, and wherein X optionally varies among polymeric or oligomeric repeats; (SEQ ID NO: 4) (d) pentapeptide Ala-Val-Gly-Val-Pro, or AVGVP; (SEQ ID NO: 5) (e) pentapeptide Ile-Pro-Gly-Val-Gly, or IPGVG; (SEQ ID NO: 6) (f) pentapeptide Leu-Pro-Gly-Val-Gly, or LPGVG; (SEQ ID NO: 7) (g) hexapeptide Val-Ala-Pro-Gly-Val-Gly, or  VAPGVG; (SEQ ID NO: 8) (h) octapeptide Gly-Val-Gly-Val-Pro-Gly-Val-Gly, or GVGVPGVG; (SEQ ID NO: 9) (i) nonapeptide Val-Pro-Gly-Phe-Gly-Val-Gly-Ala- Gly, or VPGFGVGAG; and (SEQ ID NO: 10) (j) nonapeptides Val-Pro-Gly-Val-Gly-Val-Pro-Gly- Gly, or VPGVGVPGG.

Any other polymeric or oligomeric repeat units of other sizes and constitutions can be usefully employed in the broad practice of the present invention.

In one embodiment, the ELP in the peptide active therapeutic agent-ELP construct includes repeat units of the pentapeptide Val-Pro-Gly-X-Gly, wherein X is as defined above, and wherein the ratio of Val-Pro-Gly-X-Gly pentapeptide units to other amino acid residues of the ELP is greater than about 75%, more preferably greater than about 85%, still more preferably greater than about 95%.

The peptide active therapeutic agent-ELP constructs of the invention may be synthetically, e.g., recombinantly, produced.

In the peptide active therapeutic agent-ELP construct, the ELP may be joined at a C- and/or N-terminus of the peptide active therapeutic agent, and optionally, a spacer sequence may be present separating the ELP from the peptide active therapeutic agent.

In one aspect, the invention contemplates a polynucleotide comprising a nucleotide sequence encoding a peptide active therapeutic agent-ELP fusion protein, optionally including a spacer sequence as above described, separating the ELP from the peptide active therapeutic agent. The polynucleotide may be provided as a component of an expression vector. The invention also contemplates a host cell (prokaryotic or eukaryotic) transformed by such expression vector to express the fusion protein.

The peptide active therapeutic agent-ELP construct subsequent to its synthesis or expression can be isolated by a method involving effecting a phase transition, e.g., by raising temperature, or in other manner, producing a phase transition of the fusion protein in the medium in which is contained in non-isolated form.

For example, the peptide active therapeutic agent-ELP construct may be synthesized and recovered, by steps including forming a polynucleotide comprising a nucleotide sequence encoding a peptide active therapeutic agent-ELP fusion protein exhibiting a phase transition, expressing the fusion protein in culture, and subjecting fusion protein-containing material from the culture to processing involving separation (e.g., by centrifugation, membrane separation, etc.) and inverse transition cycling to recover the peptide active therapeutic agent-ELP fusion protein.

In one specific embodiment, the peptide active therapeutic agent-ELP fusion protein includes an ELP moiety including polymeric or oligomeric repeats of a polypeptide selected from the group consisting of VPGG, IPGG, AVGVP, IPGVG, LPGVG, VAPGVG, GVGVPGVG, VPGFGVGAG, and VPGVGVPGG.

In another specific embodiment, the peptide active therapeutic agent-ELP fusion protein includes an ELP moiety including polymeric or oligomeric repeat units selected from the group consisting of LPGXG (SEQ ID NO: 11), IPGXG (SEQ ID NO: 12), and combinations thereof, wherein X is an amino acid residue that does not preclude phase transition of the ELP fusion protein.

The peptide active therapeutic agent-ELP construct of the invention comprises an amino acid sequence endowing the construct with phase transition characteristics.

The ELP in the peptide active therapeutic agent-ELP construct can include β-turn component. Examples of polypeptides suitable for use as the β-turn component are described in Urry, et al. International Patent Application PCT/US96/05186. Alternatively, the ELP in the peptide active therapeutic agent-ELP construct can be a component lacking a β-turn component, or otherwise having a different conformation and/or folding character.

The ELPs, as mentioned, can include polymeric or oligomeric repeats of various tetra-, penta-, hexa-, hepta-, octa-, and nonapeptides, including but not limited to VPGG, IPGG, VPGXG, AVGVP, IPGVG, LPGVG, VAPGVG, GVGVPGVG, VPGFGVGAG, and VPGVGVPGG (SEQ NO: 1 to SEQ NO: 10). It will be appreciated by those of skill in the art that the ELPs need not consist of only polymeric or oligomeric sequences as listed hereinabove, in order to exhibit a phase transition or otherwise constitute a suitable ELPs species for use in the peptide active therapeutic agent-ELP constructs of the invention.

In one embodiment, the peptide active therapeutic agent-ELP construct includes ELPs that are polymeric or oligomeric repeats of the pentapeptide VPGXG (SEQ ID NO: 3), where the guest residue X is any amino acid that does not eliminate the phase transition characteristics of the ELP. X may be a naturally occurring or non-naturally occurring amino acid. For example, X may be selected from the group consisting of: alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine. In a specific embodiment, X is not proline.

X may be a non-classical amino acid. Examples of non-classical amino acids include: D-isomers of the common amino acids, 2,4-diaminobutyric acid, α-amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, γ-Abu, ε-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, β-alanine, fluoro-amino acids, designer amino acids such as β-methyl amino acids, Cα-methyl amino acids, Nα-methyl amino acids, and amino acid analogs in general.

Selection of the identity of X is independent in each ELP repetition. Selection may be based on any desired characteristic, such as consideration of positively charged or negatively charged residues in the X position. It may be considered that ELPs with neutral values in the X position may have longer half-lives.

In another embodiment, the peptide active therapeutic agent-ELP construct includes ELPs that are polymeric or oligomeric repeats of the pentapeptide IPGXG (SEQ ID NO: 11) or LPGXG (SEQ ID NO: 12), where X is as defined hereinabove.

The polymeric or oligomeric repeats of the ELP sequences may be separated by one or more amino acid residues that do not eliminate the overall phase transition characteristic of the peptide active therapeutic agent-ELP construct. In one specific embodiment, when the ELP component of the peptide active therapeutic agent-ELP construct comprises polymeric or oligomeric repeats of the pentapeptide VPGXG, the ratio of VPGXG repeats to other amino acid residues of the ELP is greater than about 75%, more preferably greater than about 85%, still more preferably greater than about 95%, and most preferably greater than about 99%.

In each repeat, X is independently selected. Different resulting ELP species are distinguished here using the notation ELPk [XiYj-n], where k designates the specific type of ELP repeat unit, the bracketed capital letters are single letter amino acid codes and their corresponding subscripts designate the relative ratio of each guest residue X in the repeat units, and n describes the total length of the ELP in number of the pentapeptide repeats. For example, ELP1 [V5A2G3-10] designates a polypeptide containing 10 repeating units of the pentapeptide VPGXG, where X is valine, alanine, and glycine at a relative ratio of 5:2:3; ELP1 [K1V2F1-4] designates a polypeptide containing 4 repeating units of the pentapeptide VPGXG, where X is lysine, valine, and phenylalanine at a relative ratio of 1:2:1; ELP1 [K1V7F1-9] designates a polypeptide containing 4 repeating units of the pentapeptide VPGXG, where X is lysine, valine, and phenylalanine at a relative ratio of 1:7:1; ELP1 [V-5] designates a polypeptide containing 5 repeating units of the pentapeptide VPGXG, where X is exclusively valine; ELP1 [V-20] designates a polypeptide containing 20 repeating units of the pentapeptide VPGXG, where X is exclusively valine; ELP2 [5] designates a polypeptide containing 5 repeating units of the pentapeptide AVGVP; ELP3 [V-5] designates a polypeptide containing 5 repeating units of the pentapeptide IPGXG, where X is exclusively valine; ELP4 [V-5] designates a polypeptide containing 5 repeating units of the pentapeptide LPGXG, where X is exclusively valine.

Previous studies by Urry and colleagues have shown that the fourth residue (X) in the elastin pentapeptide sequence, VPGXG, can be altered without eliminating the formation of the β-turn. These studies also showed that the Tt is a function of the hydrophobicity of the guest residue. By varying the identity of the guest residue(s) and their mole fraction(s), ELPs can be synthesized that exhibit an inverse transition over a 0-100° C. range.

The Tt at a given ELP length can be decreased by incorporating a larger fraction of hydrophobic guest residues in the ELP sequence. Examples of suitable hydrophobic guest residues include valine, leucine, isoleucine, phenyalanine, tryptophan and methionine. Tyrosine, which is moderately hydrophobic, may also be used. Conversely, the Tt can be increased by incorporating residues, such as those selected from the group consisting of: glutamic acid, cysteine, lysine, aspartate, alanine, asparagine, serine, threonine, glysine, arginine, and glutamine; preferably selected from alanine, serine, threonine and glutamic acid.

The ELP in one embodiment is selected to provide a Tt ranging from about 10 to about 80° C., more preferably from about 35 to about 60° C., most preferably from about 38 to about 45° C.

The Tt can also be varied by varying ELP chain length. The Tt increases with decreasing MW. For polypeptides having a molecular weight >100,000, the hydrophobicity scale developed by Urry et al. (PCT/US96/05186) is preferred for predicting the approximate Tt of a specific ELP sequence.

For polypeptides having a molecular weight <100,000, the Tt is preferably determined by the following quadratic function:

Tt=M0+M1X+M2X2

where X is the MW of the FP, and M0=116.21; M1=−1.7499; M2=0.010349.

While the Tt of the ELP and, therefore of a construct of an ELP linked to a peptide active therapeutic agent, is affected by the identity and hydrophobicity of the guest residue, X, additional properties of the construct may also be affected. Such properties include, but are not limited to solubility, bioavailability or bio-unavailability, and half-life of the ELP itself and the construct.

In the Examples section below, it is seen that the ELP-coupled active therapeutic agent retains a significant amount of the therapeutic agent\'s biological activity, as compared to free protein forms of such therapeutic agent. Additionally, it is shown that ELPs exhibit long half-lives. Correspondingly, ELPs can be used in accordance with the invention to substantially increase (e.g. by greater than 10%, 20%, 30%, 50%, 100%, 200% or more, in specific embodiments) the half-life of the therapeutic agent, as conjugated with an ELP, in comparison to the half-life of the free (unconjugated) form of the therapeutic agent. Furthermore, ELPs are shown to target high blood content organs, when administered in vivo, and thus, can partition in the body, to provide a predetermined desired corporeal distribution among various organs or regions of the body, or a desired selectivity or targeting of a therapeutic agent. In sum, active ELP-therapeutic agent conjugates contemplated by the invention are administered or generated in vivo as active, site-specific compositions having extended half-lives.

In one embodiment of the invention, the ELP length is from 5 to about 500 amino acid residues, more preferably from about 10 to about 450 amino acid residues, and still more preferably from about 15 to about 150 amino acid residues. ELP length can be reduced while maintaining a target Tt by incorporating a larger fraction of hydrophobic guest residues in the ELP sequence.

The active therapeutic agent in the peptide active therapeutic agent-ELP construct can be of any suitable type. Suitable peptides include those of interest in medicine, agriculture and other scientific and industrial fields, particularly including therapeutic proteins such as erythropoietins, magainins, beta-defensins, inteferons, insulin, monoclonal antibodies, blood factors, colony stimulating factors, growth hormones, interleukins, growth factors, therapeutic vaccines, calcitonins, tumor necrosis factors (TNF), receptor antagonists, corticosteroids, and enzymes. Specific examples of such peptides include, without limitation, enzymes utilized in replacement therapy; antibacterial peptides; hormones for promoting growth; and active proteinaceous substances used in various applications. Specific examples include, but are not limited to: superoxide dismutase, interferon, asparaginease, glutamase, arginase, arginine deaminase, adenosine deaminase ribonuclease, trypsin, chromotrypsin, papin, insulin, calcitonin, ACTH, glucagon, glucagon-like peptide-1 (GLP-1), somatosin, somatropin, somatomedin, parathyroid hormone, erthyropoietin, hypothalamic releasing factors, prolactin, thyroid stimulating hormones, endorphins, enkephalins, and vasopressin.

In one embodiment of the invention, the peptide active therapeutic agent is thioredoxin.

In another embodiment, the peptide active therapeutic agent is tendamistat. The tendamistat-ELP fusion protein provides a readily-isolated, active version of tendamistat for use as an α-amylase inhibitor, e.g., in the treatment of pancreatitis. This fusion protein is suitably provided as a component of a pharmaceutical formulation in association with a pharmaceutically acceptable carrier. The tendamistat-ELP fusion protein retains most of the α-amylase inhibition activity of the free tendamistat, and is a stable construct.

In one specific embodiment, the peptide active therapeutic agent includes a physiologically active peptide selected from the group consisting of insulin, calcitonin, ACTH, glucagon, somatostatin, somatotropin, somatomedin, parathyroid hormone, erythropoietin, hypothalmic releasing factors, prolactin, thyroid stimulating hormones, endorphins, enkephalins, vasopressin, non-naturally occurring opiods, superoxide dismutase, interferon, asparaginase, arginase, arginine deaminease, adenosine deaminase ribonuclease, trypsin, chymotrypsin, and papain.

The invention thus comprehends various compositions for therapeutic (in vivo) application, wherein the peptide component of the peptide active therapeutic agent-ELP construct is a physiologically active, or bioactive, peptide. In preferred forms of such peptide-containing compositions, the coupling of the peptide component to ELP species is effected by direct covalent bonding or indirect (through appropriate spacer groups) bonding, and the peptide and ELP moieties can be structurally arranged in any suitable manner involving such direct or indirect covalent bonding, relative to one another. Thus, a wide variety of peptide species can be accommodated in the broad practice of the present invention, as necessary or desirable in a given therapeutic application.

The peptides utilized as peptide active therapeutic agents in the peptide active therapeutic agent-ELP constructs of the invention in one embodiment include enzymes utilized in replacement therapy and hormones for promoting growth. Among such enzymes are superoxide dismutase, interferon, asparaginease, glutamase, arginase, arginine deaminase, adenosine deaminase ribonuclease, cytosine deaminase, trypsin, chromotrypsin, and papin. Among the peptide hormones, specific species amenable to use in the peptide active therapeutic agent-ELP constructs of the invention include, without limitation, insulin, calcitonin, ACTH, glucagon, somatosin, somatropin, somatomedin, parathyroid hormone, erthyropoietin, hypothalamic releasing factors, prolactin, thyroid stimulating hormones, endorphins, enkephalins, and vasopressin.

In another specific aspect, the peptide active therapeutic agent in the ELPs/peptide active therapeutic agent construct is selected from among the following species, and all variants, fragments and derivatives of such species: agouti related peptide, amylin, angiotensin, cecropin, bombesin, gastrin, including gastrin releasing peptide, lactoferin, antimicrobial peptides including but not limited to magainin, urodilatin, nuclear localization signal (NLS), collagen peptide, survivin, amyloid peptides, including β-amyloid, natiuretic peptides, peptide YY, neuroregenerative peptides and neuropeptides, including but not limited to neuropeptide Y, dynorphin, endomorphin, endothelin, enkaphalin, exendin, fibronectin, neuropeptide W and neuropeptide S, peptide T, melanocortin, amyloid precursor protein, sheet breaker peptide, CART peptide, amyloid inhibitory peptide, prion inhibitory peptide, chlorotoxin, corticotropin releasing factor, oxytocin, vasopressin, cholecystokinin, secretin, thymosin, epidermal growth factor (EGF), vascular endothelial cell growth factor (VEGF), platelet-derived growth factor (PDGF), Insulin-like growth factor (IGF), fibroblast growth factors (aFGF, bFGF), pancreastatin, melanocyte stimulating hormone, osteocalcin, bradykinin, adrenomedullin, perinerin, metastatin, aprotinin, galanins, including galanin-like peptide, leptin, defensins, including but not limited to α-defensin and β-defensin, salusin, and various venoms, including but not limited to conotoxin, decorsin, kurtoxin, anenomae venom, tarantula venom; natriuretic peptides including brain natriuretic peptide (B-type natriuretic peptide, or BNP), atrial natriuretic peptide, and vasonatrin; neurokinin A, neurokinin B; neuromedin; neurotensin; orexin, pancreatic polypeptide, pituitary adenylate cyclase activating peptide (PACAP), prolactin releasing peptide, proteolipid protein (PLP), somatostatin, TNF-α; Grehlin, Protein C (Xigris), SS1(dsFv)-PE38 and pseudomonas exotoxin protein, clotting factors, including antithrombin III and Coagulation Factor VIIA, Factor VIII, Factor IX, streptokinase, tissue plasminogen activators, urokinase, beta glucocerebrosidase and alpha-D-galactosidase, alpha L-iduronidase, alpha-1,4-glucosidase, arylsulfatase B, iduronate-2-sulfatase, deoxyribunuclase I, human activated protein, follicle-stimulating hormone, chorionic gonadotropin, luteinizing hormone, somatropin, bone morphogenetic protein, nesiritide, parathyroid hormone, erythropoietin, keratinocyte growth factor, human granulocyte colony-stimulating factor (G-CSF), human granulocyte-macrophase colony stimulating factor (GM-CSF), alpha interferon, beta interferon, gamma interferon, interleukins, including IL-1, IL-1Ra, IL-2, Il-4, IL-5, IL-6, IL-10, IL-11, IL-12, glycoprotein IIB/IIIA, immune globulins, including hepatitis B, gamma globulin, venoglobulin, hirudin, aprotinin, antithrombin III, alpha-1-proteinase inhibitor, filgrastim, and etanercept.

In another embodiment, the peptide component of the peptide active therapeutic agent-ELP constructs of the present invention may be an antibody or antigen, in connection with immunotherapy, or other therapeutic intervention.

Various other proteins and peptides, such as insulin A peptide, T20 peptide, interferon alpha 2B peptide, tobacco etch virus protease, small heterodimer partner orphan receptor, androgen receptor ligand binding domain, glucocorticoid receptor ligand binding domain, estrogen receptor ligand binding domain, G protein alpha Q, 1-deoxy-D-xylulose 5-phosphate reductoisomerase peptide, G protein alpha S, angiostatin (K1-3), blue fluorescent protein (BFP), calmodulin (CalM), chloramphenicol acetyltransferase (CAT), green fluorescent protein (GFP), interleukin 1 receptor antagonist (IL-1Ra), luciferase, tissue transglutaminase (tTg), morphine modulating neuropeptide (MMN), neuropeptide Y (NPY), orexin-B, leptin, ACTH, calcitonin, adrenomedullin (AM), parathyroid hormone (PTH), defensin and growth hormone have been fused with different ELP polypeptides to form FPs that exhibit inverse phase transition behavior.

The proteins and peptides employed as active therapeutic agents can be significantly different in their primary, secondary, and tertiary structures, sizes, molecular weights, solubility, electric charge distribution, viscosity, and biological functions.

Also included within the scope of the invention are derivatives comprising FPs, which have been differentially modified during or after synthesis, e.g., by benzylation, glycosylation, acetylation, phosphorylation, amidation, PEGylation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, etc. In one embodiment, the FPs are acetylated at the N-terminus and/or amidated at the C-terminus. In another embodiment, the FPs are conjugated to polymers, e.g., polymers known in the art to facilitate oral delivery, decrease enzymatic degradation, increase solubility of the polypeptides, or otherwise improve the chemical properties of the therapeutic polypeptides for administration to humans or other animals.

The peptide active therapeutic agent-ELP constructs of the invention can be obtained by known recombinant expression techniques. To recombinantly produce the peptide active therapeutic agent-ELP construct, a nucleic acid sequence encoding the construct is operatively linked to a suitable promoter sequence such that the nucleic acid sequence encoding such fusion peptide will be transcribed and/or translated into the desired fusion peptide in the host cells. Preferred promoters are those useful for expression in E. coli, such as the T7 promoter.

Any commonly used expression system may be used, e.g., eukaryotic or prokaryotic systems. Specific examples include yeast, pichia, baculovirus, mammalian, and bacterial systems, such as E. coli, and Caulobacter.

A vector comprising the nucleic acid sequence can be introduced into a cell for expression of the peptide active therapeutic agent-ELP construct. The vector can remain episomal or become chromosomally integrated, as long as the gene carried by it can be transcribed to produce the desired RNA. Vectors can be constructed by standard recombinant DNA technology methods. Vectors can be plasmids, phages, cosmids, phagemids, viruses, or any other types known in the art, which are used for replication and expression in prokaryotic or eukaryotic cells. It will be appreciated by one of skill in the art that a wide variety of components known in the art may be included in such vectors, including a wide variety of transcription signals, such as promoters and other sequences that regulate the binding of RNA polymerase onto the promoter. Any promoter known to be effective in the cells in which the vector will be expressed can be used to initiate expression of the peptide active therapeutic agent-ELP construct. Suitable promoters may be inducible or constitutive. Examples of suitable promoters include the SV40 early promoter region, the promoter contained in the 3′ long terminal repeat of Rous sarcoma virus, the HSV-1 (herpes simplex virus-1) thymidine kinase promoter, the regulatory sequences of the metallothionein gene, etc., as well as the following animal transcriptional control regions, which exhibit tissue specificity and have been utilized in transgenic animals: elastase I gene control region which is active in pancreatic acinar cells; insulin gene control region which is active in pancreatic beta cells, immunoglobulin gene control region which is active in lymphoid cells, mouse mammary tumor virus control region which is active in testicular, breast, lymphoid and mast cells, albumin gene control region which is active in liver, alpha-fetoprotein gene control region which is active in liver, alpha 1-antitrypsin gene control region which is active in the liver, beta-globin gene control region which is active in erythroid cells, myelin basic protein gene control region which is active in oligodendrocyte cells in the brain, myosin light chain-2 gene control region which is active in skeletal muscle, and gonadotropin releasing hormone gene control region which is active in the hypothalamus.

In one embodiment, a mammal is genetically modified to produce the peptide active therapeutic agent-ELP construct in its milk. Techniques for performing such genetic modifications are described in U.S. Pat. No. 6,013,857, issued Jan. 11, 2000, for “Transgenic Bovines and Milk from Transgenic Bovines.” The genome of the transgenic animal is modified to comprise a transgene comprising a DNA sequence encoding a peptide active therapeutic agent-ELP construct operably linked to a mammary gland promoter. Expression of the DNA sequence results in the production of the peptide active therapeutic agent-ELP construct in the milk. The peptide active therapeutic agent-ELP construct can then be isolated by phase transition from milk obtained from the transgenic mammal. The transgenic mammal is preferably a bovine.

The peptide active therapeutic agent-ELP constructs of the invention can be separated from other contaminating proteins to high purity using inverse transition cycling procedures, e.g., utilizing the temperature-dependent solubility of the peptide active therapeutic agent-ELP construct, or salt addition to the medium containing the construct. Successive inverse phase transition cycles can be used to obtain a high degree of purity.

In addition to temperature and ionic strength, other environmental variables useful for modulating the inverse transition of peptide active therapeutic agent-ELP constructs include pH, the addition of inorganic and organic solutes and solvents, side-chain ionization or chemical modification, and pressure.

In one specific illustrative embodiment of the invention, a 10 polypentapeptide ELP (an ELP 10-mer) is constructed. The ELP 10-mer may be oligomerized or polymerized up to 18 times to create a library of ELPs with precisely specified molecular masses (10-, 20-, 30-, 60-, 90-, 120-, 150-, and 180-mers). The ELP polymers or oligomers may then be fused to the C- or N-terminus of the peptide active therapeutic agent, to form the peptide active therapeutic agent-ELP construct. A second peptide active therapeutic agent may be fused to the ELP component of the fusion protein construct, providing a ternary fusion. Optionally, one or more spacers may be used to separate the ELP component from the peptide active therapeutic agent(s).

The invention thus affords a peptide active therapeutic agent-ELP construct in which the peptide active therapeutic agent may be a natural or synthetic version of any of a wide variety of endogenous molecules, or alternatively a non-naturally-occurring peptide species, or a functional equivalent of any of the foregoing.

The peptide active therapeutic agent-ELP constructs of the invention overcome the major deficiency of peptide active therapeutic agents when given parenterally, namely, that such peptides are easily metabolized by plasma proteases. The oral route of administration of peptide active therapeutic agents is even more problematic because in addition to proteolysis in the stomach, the high acidity of the stomach destroys such peptide active therapeutic agents before they reach their intended target tissue. Peptides and peptide fragments produced by the action of gastric and pancreatic enzymes are cleaved by exo and endopeptidases in the intestinal brush border membrane to yield di- and tripeptides, and even if proteolysis by pancreatic enzymes is avoided, polypeptides are subject to degradation by brush border peptidases. Any of the peptide active therapeutic agents that survive passage through the stomach are further subjected to metabolism in the intestinal mucosa where a penetration barrier prevents entry into the cells. The peptide active therapeutic agent-ELP constructs of the invention overcome such deficiencies, and provide compositional forms of the peptide active therapeutic agent having enhanced efficacy, in bioavailability, bio-unavailability, therapeutic half-life, degradation assistance, etc.

The peptide active therapeutic agent-ELP constructs of the invention thus enable oral and parenteral dose forms, as well as various other dose forms, by which peptide active therapeutic agents can be utilized in a highly effective manner. For example, such constructs enable dose forms that achieve high mucosal absorption, and the concomitant ability to use lower doses to elicit an optimum therapeutic effect.

The ELP/peptide active therapeutic agent construct may also include a spacer as a moiety in the construct. The spacer may be of any suitable type, and may be a peptide spacer, or alternatively a non-peptide chemical moiety.

Peptide spacers may be protease-cleavable or non-cleavable. By way of example, cleavable peptide spacer species include, without limitation, in a peptide sequences recognized by proteases of varying type, such as thrombin, factor Xa, plasmin (blood proteases), metalloproteases, cathepsins (e.g., GFLG, etc.), and proteases found in other corporeal compartments. The non-cleavable spacer may likewise be of any suitable type, including, for example, non-cleavable spacer moieties having the formula [(Gly)n-Ser]m where n is from 1 to 4, inclusive, and m is from 1 to 4, inclusive.

Non-peptide chemical spacers can additionally be of any suitable type, including for example, by functional linkers described in Bioconjugate Techniques, Greg T. Hermanson, published by Academic Press, Inc., 1995, and those specified in the Cross-Linking Reagents Technical Handbook, available from Pierce Biotechnology, Inc. (Rockford, Ill.), the disclosures of which are hereby incorporated by reference, in their respective entireties. Illustrative chemical spacers include homobifunctional linkers that can attach to amine groups of Lys, as well as heterobifunctional linkers that can attach to Cys at one terminus, and to Lys at the other terminus, and other bifunctional linkers that can link proteins to the Fc region of antibodies, in which the antibody\'s carbohydrate is first oxidized to a diol or aldehyde.

The peptide active therapeutic agent-ELP constructs of the invention have application in prophylaxis or treatment of condition(s) or disease state(s). Although such constructs are described herein with reference to peptide active therapeutic agents having utility for animal subjects, the invention also contemplates peptide active therapeutic agent-ELP constructs having utility for prophylaxis or treatment of condition(s) or disease state(s) in plant systems. By way of example, the peptide component of the peptide active therapeutic agent-ELP construct having such plant utility may have insecticidal, herbicidal, fungicidal, and/or pesticidal efficacy.

A further aspect of the invention relates to gene therapy utilizing fusion gene therapeutic compositions of the invention, in conjunction with vectors of any suitable type, e.g., AAV, vaccinia, pox virus, HSV, retrovirus, lipofection, RNA transfer, etc.

In therapeutic usage, the present invention contemplates a method of treating an animal subject having or latently susceptible to such condition(s) or disease state(s) and in need of such treatment, including administering to such animal an effective amount of a peptide active therapeutic agent-ELP construct of the present invention which is therapeutically effective for said condition or disease state.

Animal subjects to be treated by the peptide active therapeutic agent-ELP constructs of the present invention include both human and non-human animal (e.g., bird, dog, cat, cow, horse) subjects, and preferably are mammalian subjects, and most preferably human subjects.

Depending on the specific condition or disease state to be combated, animal subjects may be administered peptide active therapeutic agent-ELP constructs of the invention at any suitable therapeutically effective and safe dosage, as may readily be determined within the skill of the art, without undue experimentation, based on the disclosure herein.

In general, suitable doses of the peptide active therapeutic agent in the peptide active therapeutic agent-ELP construct for achievement of therapeutic benefit, can for example be in a range of 1 microgram (μg) to 100 milligrams (mg) per kilogram body weight of the recipient per day, preferably in a range of 10 μg to 50 mg per kilogram body weight per day and most preferably in a range of 10 μg to 50 mg per kilogram body weight per day. The desired dose can be presented as two, three, four, five, six, or more sub-doses administered at appropriate intervals throughout the day. These sub-doses can be administered in unit dosage forms, for example, containing from 10 μg to 1000 mg, preferably from 50 μg to 500 mg, and most preferably from 50 μg to 250 mg of active ingredient per unit dosage form. Alternatively, if the condition of the recipient so requires, the doses may be administered as a continuous infusion.

The mode of administration and dosage forms will of course affect the therapeutic amount of the peptide active therapeutic agent that is desirable and efficacious for a given treatment application.

For example, orally administered dosages can be at least twice, e.g., 2-10 times, the dosage levels used in parenteral administration methods, for the same peptide active therapeutic agent.

The peptide active therapeutic agent-ELP constructs of the invention may be administered per se as well as in forms of such constructs including pharmaceutically acceptable esters, salts, and other physiologically functional derivatives thereof.

The present invention also contemplates pharmaceutical formulations, both for veterinary and for human medical use, which include peptide active therapeutic agent-ELP constructs of the invention.

In such pharmaceutical and medicament formulations, the peptide active therapeutic agent-ELP construct can be utilized together with one or more pharmaceutically acceptable carrier(s) therefore and optionally any other therapeutic ingredients. The carrier(s) must be pharmaceutically acceptable in the sense of being compatible with the other ingredients of the formulation and not unduly deleterious to the recipient thereof. The peptide active therapeutic agent-ELP construct is provided in an amount effective to achieve the desired pharmacological effect, as described above, and in a quantity appropriate to achieve the desired daily dose.

The formulations of the peptide active therapeutic agent-ELP constructs include those suitable for parenteral as well as non-parenteral administration, and specific administration modalities include oral, rectal, buccal, topical, nasal, ophthalmic, subcutaneous, intramuscular, intravenous, transdermal, intrathecal, intra-articular, intra-arterial, sub-arachnoid, bronchial, lymphatic, vaginal, and intra-uterine administration. Formulations suitable for oral and parenteral administration are preferred.